摘要Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production of α-LA.It was found that α-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only produced α-LA intracellular,but not extracellular.To improve the expression and secretion of α-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(PaprE,P43,and Pglv)were compared and applied.The results indicated that the strain RIK1285-pBE-Pglv-YjcN-LALBA had the highest α-LA yield,reaching 122.04 μg/mL.This study demonstrates successful expression and secretion of human α-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.